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Dissertation zugänglich unter
Identifikation von Resistenzmechanismen gegenüber EGFR-Tyrosinkinase Inhibitoren in EGFR-amplifizierten und EGFRvIII-positiven Glioblastomen
Mechanisms of EGFR-tyrosinkinase-inhibitor-resistance of EGFR-amplified and EGFRvIII-positive glioblastoma
Dokument 1.pdf (8.479 KB)
Epidermaler Wachstumsfaktor-Rezeptor , Glioblastom
Freie Schlagwörter (Deutsch):
EGFRvIII , PI3K , Erlotinib , Histondeacetylase-Inhibitor , Tyrosinkinase-Inhibitor
Lamszus, Katrin (Prof. Dr.)
Tag der mündlichen Prüfung:
Kurzfassung auf Englisch:
Glioblastoma is the most frequent, aggressive and maligne human brain tumor. One of the most common characteristic aberration of primary GBM is the overexpression of EGFR which is caused by increased translation and/or an amplification of the EGFR gene and is often associated with the expression of the constitutive active deletion variant EGFRvIII. In the past few years different EGFR-specific therapies such as EGFR specific tyrosine kinase inhibitors (TKI) and antibodies (mAB) were developed. Because glioblastoma patients do often not respond or develop resistance against these therapeutics, initial positive preclinical results could not be confirmed in clinical studies. In the present study the impact of wtEGFR and EGFRvIII on GBM initiation as well as the influence of EGFR amplification and EGFRvIII expression on the efficacy of EGFR-specific therapies and the development of TKI resistance was analyzed using EGFR amplified and EGFRvIII expressing GBM model cell lines.
Using in vivo xenograft experiments with individually fluorescence-labeled BS153 cells (RGB marking), it was demonstrated that in contrast to U87MG tumors, which showed no EGFR amplification and were EGFRvIII negative, BS153 tumors were only induced by a limited number of EGFR positive clones (< 8) with tumor initiation capacity. This supports the thesis of cancer stem cells and highlights the impact of EGFR on cancer stem cells.
Treatment of BS153 cells with the TKIs erlotinib and gefitinib induced a significant reduction of cell proliferation and migration whereas mAB cetuximab showed no inhibitory effect. Chronical treatment of BS153 cells with erlotinib, led to erlotinib resistant cell line (BS153resE), which showed increased EGFRvIII protein expression and reduction of wtEGFR (mRNA and protein) compared to the parental cell line. In contrast, the EGFR amplification of BS153resE cells was not changed in comparison to BS153 cells. EGFRvIII knock down experiments with BS153resE cells showed, that EGFRvIII contributes to the regulation of the PI3K Akt and the Ras Raf MEK Erk signaling pathway. The central importance of EGFRvIII for BS153resE cell was further elucidated by the observation that the knock down of EGFRvIII caused a resensitization to erlotinib.
In in vivo experiments with immune deficient mice, BS153 and BS153resE cells were tumorigenic and induced invasive tumors, whereas the wtEGFR/EGFRvIII phenotype was preserved. Chronic erlotinib treatment of RGB labeled BS153 cells showed, that the erlotinib resistant cell line BS153resE emerged from parental BS153 cell by a modification of gene expression and not by clonal selection. Besides a modified wtEGFR/EGFRvIII status, BS153resE cells showed increased expression of the catalytic p110δ subunit of PI3K and decreased expression of PTEN, the most important negative regulator of the PI3K-Akt signaling pathway. The knock down of PI3K(p110δ) caused a decrease cell proliferation and a resensitization of BS153resE cells to Erlotinib. Because PI3K(p110δ) is not expressed by healthy brain tissue, the inhibition of PI3K(p110δ) is an interesting and promising new treatment approach to overcome acquired erlotinib resistance.
One possibility to increase the efficacy of EGFR targeted therapy is the modulation of EGFR expression by epigenetic mechanisms. The present study demonstrates that the treatment of BS153 and BS153resE cells with histone deacetylase inhibitors (HDACi) results in reduced wtEGFR and EGFRvIII expression as well as decreased proliferation, whereas cell migration is not affected by HDACi treatment. Importantly, the simultaneous inhibition of EGFR and HDAC activity by Erlotinib and HDACi caused an additive inhibition of BS153 and BS153resE proliferation. Therefore, it was possible to sensitize BS153resE cell to Erlotinib by parallel treatment with HDAC and the TKI.
In summary, the upregulation of EGFRvIII and PI3K(p110δ) seem to be central mechanisms of the erlotinib resistance of EGFR amplified and EGFRvIII expressing GBM cells. Furthermore, the inhibition of PI3K(p110δ) as well as the epigenetic modulation of wtEGFR/EGFRvIII expression by HDACi are promising treatment approaches to prevent and overcome erlotinib resistance.